Metabolism in Action: Quantitative Fluxes in Mammals Abstract The molecular connections involved in metabolism are the best understood of any major biochemical network. Nevertheless, metabolic disease remains at epidemic levels, and other diseases involving aberrant metabolism, such as cancer, continue unabated. A key step towards addressing these major unmet medical needs is to understand the integrated activity of metabolic pathways, and their modulation by diet and disease. Despite the recent revitalized interest in metabolism, systems-level methods for measuring metabolic activity in intact mammals remain in their infancy.Here I propose to combine isotope tracing, state-of-the-art metabolomics technology, and computational modeling to reveal metabolic activity at the whole body level. Labeled nutrients will be infused intravenously into mice, tissues sampled, and metabolite labeling quantified by liquid chromatography-mass spectrometry. Metabolic pathway flows (fluxes) consistent with the tracer data will be identified within the context of whole body metabolic model, which encompasses tissue-specific metabolic activity and exchange of metabolites between organs via the circulation. The fluxes will also be constrained by macroscopic measurements like food and oxygen uptake and carbon dioxide and urea excretion rates, tying our approach to classical physiological measurements. Through these studies, we will revisit from a quantitative perspective the overall operation of mammalian metabolism. Anticipated outcomes include enhanced understanding of the sources and consumption routes of circulating metabolites, discovery of novel metabolic cycles connecting different organs, and quantitative measurement methods of broad utility for probing disease models. Application of these methods will reveal disease-specific pathway dysregulation. The overall impact will be a more holistic and comprehensive understanding of metabolism that enables rational dietary guidance and therapeutic intervention across a broad spectrum of diseases.